CN110714090A - Kit for detecting free nucleic acid of blood stream infection pathogen in blood plasma - Google Patents
Kit for detecting free nucleic acid of blood stream infection pathogen in blood plasma Download PDFInfo
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Abstract
The application belongs to the technical field of biology and discloses a kit and a detection method for detecting free nucleic acid of a blood stream infection pathogen in blood plasma. The kit for detecting the free nucleic acid of the blood stream infection pathogen in the plasma comprises a primer group for amplifying a target sequence of the free DNA of the blood stream infection pathogen and a detection probe. The kit has good specificity for free nucleic acid DNA of blood stream infection pathogens including Klebsiella pneumoniae, Aspergillus flavus and Pseudomonas aeruginosa in a plasma sample, and can realize rapid, accurate and sensitive identification of the blood stream infection pathogens including Klebsiella pneumoniae, Aspergillus flavus and Pseudomonas aeruginosa in the plasma sample. The kit and the detection method are simple and convenient to operate, and have the advantages of good detection specificity, high sensitivity and wide detection range. Experiments show that the lowest detection limits of the kit for detecting klebsiella pneumoniae, aspergillus flavus and pseudomonas aeruginosa are 0.2, 0.4 and 0.3CFU/ml respectively.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit for detecting free nucleic acid of a blood stream infection pathogen in blood plasma, in particular to a kit for detecting the free nucleic acid of the blood stream infection pathogen in the blood plasma by applying a QPCR (quick-quench polymerase chain reaction) technology.
Background
Bloodstream infection (BSI) is one of clinically serious infectious diseases, and can be classified into sepsis, sepsis and septic shock according to symptoms. The bloodstream infection is caused by the invasion of pathogenic bacteria into the blood system, the growth and the reproduction of pathogenic bacteria in the blood, and a large amount of toxins are generated to further cause acute systemic infection, so that the morbidity and the mortality are high, and common infection sources comprise bacteria and fungi.
At present, a rapid and accurate detection method aiming at blood stream infection is lacked. The gold standard for diagnosis of bloodstream infections is blood culture, and determination of pathogens by biochemical or microscopic observation, but this method is long, usually takes several days to determine the results, and for pathogens that are difficult or impossible to culture, it is difficult to detect accurately. In the absence of accurate diagnosis, clinicians may blindly use antibiotics to alleviate the condition, resulting in decreased survival of patients, emergence of bacterial resistance, and increased treatment costs, among other things.
In the early stage of disease infection, the early identification of pathogens is the key for effective treatment, in the existing detection kit on the market, the pathogens are detected by a blood culture method, the detection time is usually over 24 hours, meanwhile, the abuse influence of antibacterial drugs is avoided, the culture detection rate is greatly reduced, and the clinical diagnosis and treatment of serious and urgent blood stream infection can not be met.
The molecular detection technology can be applied to the detection of blood stream infection pathogens. However, because the content of pathogenic microorganisms in blood is low, the detection of the DNA of the pathogenic microorganisms is interfered by a large amount of red blood cells, heme, white blood cells, human genomes and the like in a whole blood sample, and the detection sensitivity is seriously influenced. The products on the market of Biofire company for detecting the blood stream infection pathogen adopt a molecular detection technology, are only used for detecting the pathogenic microorganisms of a cultured positive culture, cannot directly detect from original whole blood, and can detect only by blood culture, so that the product application is greatly limited.
Aiming at the clinical blood stream infected patients, the content of pathogenic bacteria in blood samples is not more than 10CFU/mL, or even lower. The clinical need is a new technology for detecting blood stream infection pathogens, which can overcome the interference of a large amount of red blood cells, heme, white blood cells and human genomes in whole blood samples, can detect the pathogens causing blood stream infection quickly with high sensitivity, guides the selection and use of antibiotics, reduces blind medication and shortens the treatment time of patients.
Disclosure of Invention
In view of the above, the present invention provides a method for detecting blood stream infectious pathogens in plasma by applying QPCR technology and a corresponding detection kit, and the kit and the detection method can accurately, rapidly and sensitively identify blood stream infectious pathogens.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a kit for detecting free nucleic acid of blood stream infection pathogen in plasma comprises a primer group for amplifying target sequence of free DNA of blood stream infection pathogen and a detection probe.
The kit can adopt QPCR technology to adopt plasma as a detection sample, adopts free nucleic acid DNA of blood stream infection pathogens as a detection marker to detect the free nucleic acid DNA of the blood stream infection pathogens in the plasma, is not genome DNA, and can avoid the interference of a large number of red blood cells, heme, white blood cells and human genome groups in a whole blood sample on the detection of the blood stream infection pathogens. The kit can identify the pathogen type of blood stream infection by extracting the free nucleic acid DNA of the pathogen in the plasma and detecting the free nucleic acid DNA characteristic of the pathogen.
In the invention, the blood stream infection pathogen is at least one of klebsiella pneumoniae, aspergillus flavus and pseudomonas aeruginosa.
In some embodiments, the bloodstream infection pathogen is klebsiella pneumoniae, aspergillus flavus, and pseudomonas aeruginosa.
In the present invention, the target segment sequence is a high abundance target sequence.
In some embodiments, the kit, in,
the sequence of the amplification primer group of the target sequence of the Klebsiella pneumoniae free DNA is shown as SEQ ID NO: 1 and 2, the sequence of the detection probe is shown as SEQ ID NO: 3 is shown in the specification;
the sequence of the amplification primer group of the target sequence of the aspergillus flavus free DNA is shown as SEQ ID NO: 4 and 5, the sequence of the detection probe is shown as SEQ ID NO: 6 is shown in the specification;
the sequence of the amplification primer group of the target sequence of the pseudomonas aeruginosa free DNA is shown as SEQ ID NO: 7 and 8, the sequence of the detection probe is shown as SEQ ID NO: shown at 9.
In the present invention, the kit may further comprise a primer set and/or a detection probe for amplifying the internal standard fragment.
In some embodiments, the sequence of the internal standard fragment is as set forth in SEQ ID NO: 10(5 '-3' gatgaagttg gtggtgaggccctgggcaggttggtatcaaggttacaagacaggtttaaggagaccaatagaaactgggca).
In some embodiments, the sequence of the primer set for amplifying the internal standard fragment is as shown in SEQ ID NO: 11 and 12, the sequence of the detection probe is shown as SEQ ID NO: shown at 13.
Further, in the present invention, the kit may further include at least one of a plasma nucleic acid free DNA extraction reagent, a PCR reaction buffer, deoxyribonucleoside triphosphates, a positive control, a negative control, and an enzyme mixture.
The reagent for extracting the free DNA of the plasma nucleic acid can be used for extracting the free DNA of the pathogen in the plasma. As will be understood by those skilled in the art, the nucleic acid free DNA extraction reagent may be prepared by itself according to the existing formulation, or the nucleic acid free DNA extraction kit may be used to extract free nucleic acid DNA in advance. Such as QIAamp Circulating nucleic acid Kit.
The PCR buffer solution is a common reagent for PCR reaction, mainly comprises a buffer salt solution, a surfactant, a PCR enhancer and the like, and can be prepared according to the existing formula. In some embodiments, the PCR reaction buffer is formulated with 50mM Tirs-HCl, 100mM KCl, 2.5mM MgCl2, 1mM DTT, 0.04% Tween-20, 3% trehalose.
In the invention, the enzyme mixed solution in the kit consists of heat-resistant DNA polymerase and uracil DNA glycosylase.
The invention also provides a method for detecting the free nucleic acid of the blood stream infection pathogen in the plasma, which takes the plasma as a detection sample, the DNA of the free nucleic acid of the blood stream infection pathogen as a detection marker, and carries out QPCR detection on the free nucleic acid of the blood stream infection pathogen in the plasma by using a primer group and a detection probe of a target sequence of the DNA of the blood stream infection pathogen.
The detection method can avoid the interference of a large number of red blood cells, heme, white blood cells and human genome in a whole blood sample on the detection of the blood stream infection pathogens, and detect the free nucleic acid DNA of the pathogens by extracting the free nucleic acid DNA of the pathogens in the blood plasma, thereby identifying the types of the pathogens of the blood stream infection. The detection method adopts a QPCR method instead of a sequencing method, and is convenient for clinical application.
According to the technical scheme, the invention provides a kit and a detection method for detecting free nucleic acid of blood stream infection pathogens in blood plasma by applying QPCR technology. The kit for detecting the free nucleic acid of the blood stream infection pathogen in the plasma comprises a primer group for amplifying a target sequence of the free DNA of the blood stream infection pathogen and a detection probe. The kit provided by the invention has good specificity for free nucleic acid DNA of blood stream infection pathogens including Klebsiella pneumoniae, Aspergillus flavus and Pseudomonas aeruginosa in a plasma sample, can realize rapid, accurate and sensitive identification of blood stream infection pathogens including Klebsiella pneumoniae, Aspergillus flavus and Pseudomonas aeruginosa in the plasma sample by combining a QPCR detection method, provides a reliable experimental basis for diagnosing nucleic acid of Klebsiella pneumoniae, Aspergillus flavus and Pseudomonas aeruginosa, and solves the technical problems of low efficiency, poor specificity and low sensitivity of the existing kit. The kit and the detection method are simple and convenient to operate, and have the advantages of good detection specificity, high sensitivity and wide detection range. Experiments show that the lowest detection limits of the kit for detecting klebsiella pneumoniae, aspergillus flavus and pseudomonas aeruginosa are 0.2CFU/ml, 0.4CFU/ml and 0.3CFU/ml respectively.
Detailed Description
The invention discloses a kit for detecting free nucleic acid of blood stream infection pathogens in blood plasma. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, the reagents and instruments used in the examples of the present invention are commercially available products, and are commercially available.
Example 1 extraction of free nucleic acids from blood stream infectious pathogens from plasma samples
Collecting 1.2ml of human plasma samples treated by K2EDTA by using a sterile 5ml centrifuge tube, extracting by adopting a QIAamp Circulating Nucleic Acid Kit of Kejie company, carrying out the operation steps according to the instruction, and storing the extracted Nucleic Acid below-75 ℃ for later use.
Example 2 primer and Probe optimization experiment
Because free DNA is short fragment, different target sequences have different abundances in plasma. Different target gene sequences and different amplification fragment lengths are designed aiming at Klebsiella pneumoniae, Aspergillus flavus and Pseudomonas aeruginosa, the plasma of a positive patient is adopted to extract free DNA, and the primers and the probes are screened.
TABLE 1 screening sequences for primers and probes
For the same sample, the smaller the detection Ct value is, the higher the detection sensitivity of the combination of the primer and the probe is. The results in Table 1 identify the primers and probes with high sensitivity, and the sequences are shown in Table 2.
TABLE 2 high sensitivity primer and probe sequences
Example 3 preparation of Triplex nucleic acid detection kit for Klebsiella pneumoniae, Aspergillus flavus and Pseudomonas aeruginosa
The sequences of the primers and probes in the kit are shown in Table 2.
The kit also comprises an internal standard primer and an internal standard probe, and the sequence is as follows:
an internal standard forward primer: 5 '-3' gatgaagttggtggtgaggcc (SEQ ID NO: 11);
internal standard reverse primer: 5 '-3' tgcccagtttctattggtctcc (SEQ ID NO: 12);
internal standard probe: 5 '-3' gttccaccagaaaaaggaaccagacagg (SEQ ID NO: 13);
the kit also comprises: 10mM dNTPs, 5U/. mu.l Taq enzyme, 50mM MgCl2. The kit also includes a negative control (sterile water) and a positive control (artificially synthesized at a concentration of 1X 10)6Pseudovirus of Copies/ml).
Example 4 detection method of the invention
The detection method of the invention is Real time QPCR, and the composition of each detection system is shown in Table 3.
TABLE 3 detection System
| Composition (I) | Dosage of |
| 10mM dNTPs | 2μl |
| 5U/. mu.l Taq enzyme | 0.5μl |
| 50mM MgCl2 | 1.5μl |
| 20mM Klebsiella pneumoniae forward primer | 1μl |
| 20mM Klebsiella pneumoniae reverse primer | 1μl |
| 20mM Klebsiella pneumoniae probe | 0.5μl |
| 20mM Aspergillus flavus forward primer | 1μl |
| 20mM Aspergillus flavus reverse primer | 1μl |
| 20mM Aspergillus flavus probe | 0.5μl |
| 20mM Pseudomonas aeruginosa forward primer | 1μl |
| 20mM Pseudomonas aeruginosa reverse primer | 1μl |
| 20mM Pseudomonas aeruginosa probe | 0.5μl |
| 20mM internal standard forward primer | 0.7μl |
| 20mM internal standard reverse primer | 0.7μl |
| 20mM internal standard probe | 0.4μl |
| Sterilized purified water | Make up to 25. mu.l |
Fluorescence detection channel selection:
(1) selecting a FAM channel (ReporTer: FAM, Quencher: none), and detecting Klebsiella pneumoniae;
(2) selecting a ROX channel (ReporTer: ROX, Quencher: none), and detecting aspergillus flavus;
(3) selecting a CY5 channel (ReporTer: CY5, Quencher: none), and detecting pseudomonas aeruginosa;
(4) selecting an HEX channel, and detecting an internal standard;
(5) the ReferenCe fluorescence (PAStive ReferenCe) was set to none.
After the reaction is finished, the fluorescence quantitative PCR instrument automatically stores the result, the QPCR data analysis software carried by the fluorescence quantitative PCR instrument is used for carrying out automatic analysis or manually adjusting the starting value, the ending value and the threshold line value of the baseline for analysis, and then the CT value and the fixed value result of the sample are recorded. The specific test results were analyzed as follows:
the probe channels shown in SEQ ID NO.3, SEQ ID NO.6 and SEQ ID NO.9 have no fluorescence value, the CT value of the probe channel shown in SEQ ID NO.13 is less than or equal to 35, and the report detection result is negative;
the CT value of the probe channel shown by SEQ ID NO.3 is less than or equal to 38, but the CT values of the probe channels shown by SEQ ID NO.6 and SEQ ID NO.9 are more than or equal to 38, and the CT value of the probe channel shown by SEQ ID NO.13 is less than or equal to 35, and the result is reported to be positive by Klebsiella pneumoniae;
the CT values of the probe channels shown by SEQ ID NO.6 are all less than or equal to 38, but the CT values of the probe channels shown by SEQ ID NO.3 and SEQ ID NO.9 are more than or equal to 38, and the CT value of the probe channel shown by SEQ ID NO.13 is less than or equal to 35, and the positive Aspergillus flavus is reported;
the CT values of the probe channels shown by SEQ ID NO.9 are all less than or equal to 38, but the CT values of the probe channels shown by SEQ ID NO.3 and SEQ ID NO.6 are more than or equal to 38, and the report shows that the probe channels are positive to pseudomonas aeruginosa;
when the CT value of the probe channel shown in SEQ ID NO.13 is more than 35, and the negative control has the CT value or presents a typical S amplification curve, and the positive control has no CT value or no amplification curve, the detection result is invalid, the reason should be searched and eliminated, and the test is repeated.
Example 5 feasibility test of the kit of the invention
1. Limit of detection (LOD) test
(1) Preparing a triple nucleic acid detection reagent for Klebsiella pneumoniae, Aspergillus flavus and Pseudomonas aeruginosa: the method of example 3 is adopted to prepare the triple nucleic acid detection reagent for Klebsiella pneumoniae, Aspergillus flavus and Pseudomonas aeruginosa.
(2) Extraction of free nucleic acids of blood stream infection pathogens in plasma samples: nucleic acid was extracted from the sample by the method of example 1 for use.
(3) Sample detection
Adding 25 mul of the treated specimen supernatant into a triple nucleic acid detection reagent reaction tube for Klebsiella pneumoniae, Aspergillus flavus and Pseudomonas aeruginosa, wherein each concentration is 20 multiple holes, and simultaneously adding 25 mul of purified water into the detection solution as a negative control, and detecting according to the detection method of the embodiment 4.
(4) Analysis of results
By adopting the kit prepared in the embodiment 3 and the detection method in the embodiment 4 to detect samples with various concentration gradients of the Klebsiella pneumoniae, Aspergillus flavus and Pseudomonas aeruginosa triple nucleic acid detection reagent, the results show that the detection sensitivity of the detection method is as follows: LOD Klebsiella pneumoniae is 0.2CFU/ml, Aspergillus flavus is 0.4CFU/ml, and Pseudomonas aeruginosa is 0.3 CFU/ml. The specific data are shown in tables 5, 6 and 7.
TABLE 5 confirmation of detection limits of Klebsiella pneumoniae
| Sample concentration (CFU/ml) | Detecting the number of repetitions | Number of positive tests | Rate of positive detection |
| 0 | 21 | 0 | 0.00％ |
| 0.075 | 21 | 16 | 76.19％ |
| 0.2 | 21 | 21 | 100.00％ |
| 0.4 | 21 | 21 | 100.00％ |
| 0.8 | 21 | 21 | 100.00％ |
TABLE 6 Aspergillus flavus detection Limit confirmation
| Sample concentration (CFU/ml) | Detecting the number of repetitions | Number of positive tests | Rate of positive detection |
| 0 | 21 | 0 | 0.00％ |
| 0.15 | 21 | 15 | 71.43％ |
| 0.4 | 21 | 21 | 100.00％ |
| 0.8 | 21 | 21 | 100.00％ |
| 1.6 | 21 | 21 | 100.00％ |
TABLE 7 Pseudomonas aeruginosa detection Limit validation
| Sample concentration (CFU/ml) | Detecting the number of repetitions | Number of positive tests | Rate of positive detection |
| 0 | 21 | 0 | 0.00％ |
| 0.12 | 21 | 14 | 66.67％ |
| 0.3 | 21 | 21 | 100.00％ |
| 0.6 | 21 | 21 | 100.00％ |
| 1.2 | 21 | 21 | 100.00％ |
2. Cross-reactive conditions with other diseases
(1) Preparing a triple nucleic acid detection reagent for Klebsiella pneumoniae, Aspergillus flavus and Pseudomonas aeruginosa: the method of example 3 is adopted to prepare the triple nucleic acid detection reagent for Klebsiella pneumoniae, Aspergillus flavus and Pseudomonas aeruginosa.
(2) Cross pathogen sample extraction
The nucleic acid in the sample is extracted from staphylococcus aureus, cryptococcus neoformans, corynebacterium diphtheriae, haemophilus influenzae, bacillus denaturans, streptococcus pneumoniae, escherichia coli, rhizopus oryzae, candida albicans and candida albicans in the tube by the method of example 1 for standby.
(3) Sample detection
Adding 25 mul of the treated specimen supernatant into a triple nucleic acid detection reagent reaction tube for Klebsiella pneumoniae, Aspergillus flavus and Pseudomonas aeruginosa, simultaneously adding 25 mul of purified water into the detection solution as a negative control, extracting Klebsiella pneumoniae, Aspergillus flavus and Pseudomonas aeruginosa as a positive control test, and detecting according to the detection method in the embodiment 4.
(4) Analysis of results
By using the kit prepared in example 3 and the detection method of example 4 to detect pathogens other than klebsiella pneumoniae, aspergillus flavus and pseudomonas aeruginosa, the results show that: the kit disclosed by the invention has positive control detection results on Klebsiella pneumoniae, Aspergillus flavus and Pseudomonas aeruginosa, has negative control detection results, has no cross reaction with pathogen infection samples such as staphylococcus aureus, cryptococcus neoformans, Corynebacterium diphtheriae, Haemophilus influenzae, Bacillus denaturans, Streptococcus pneumoniae, Escherichia coli, Rhizopus oryzae, Candida albicans and Candida albicans, and has high specificity. The specific results are shown in Table 8.
TABLE 8 Cross-reaction experiments
| Sample name | The result of the detection | Sample name | The result of the detection |
| Klebsiella pneumoniae | Positive for | Modified bacillus | Negative of |
| Aspergillus flavus | Positive for | Streptococcus pneumoniae | Negative of |
| Pseudomonas aeruginosa | Positive for | Escherichia coli | Negative of |
| Staphylococcus aureus | Negative of | Rhizopus oryzae | Negative of |
| Cryptococcus neoformans | Negative of | Candida albicans | Negative of |
| Diphtheria bacillus | Negative of | Candida albicans | Negative of |
| Haemophilus influenzae | Negative of | Negative control | Negative of |
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Sequence listing
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Claims (10)
1. A kit for detecting free nucleic acid of blood stream infection pathogen in plasma comprises a primer group for amplifying target sequence of free DNA of blood stream infection pathogen and a detection probe.
2. The kit of claim 1, wherein the bloodstream infection pathogen is at least one of klebsiella pneumoniae, aspergillus flavus, pseudomonas aeruginosa.
3. The kit of claim 1, wherein the target segment sequence is a high abundance target sequence.
4. The kit according to claim 2 or 3,
the sequence of the amplification primer group of the target sequence of the Klebsiella pneumoniae free DNA is shown as SEQ ID NO: 1 and 2, the sequence of the detection probe is shown as SEQ ID NO: 3 is shown in the specification;
the sequence of the amplification primer group of the target sequence of the aspergillus flavus free DNA is shown as SEQ ID NO: 4 and 5, the sequence of the detection probe is shown as SEQ ID NO: 6 is shown in the specification;
the sequence of the amplification primer group of the target sequence of the pseudomonas aeruginosa free DNA is shown as SEQ ID NO: 7 and 8, the sequence of the detection probe is shown as SEQ ID NO: shown at 9.
5. The kit according to any one of claims 1 to 4, further comprising a primer set and/or a detection probe for amplifying an internal standard fragment.
6. The kit according to claim 5, wherein the sequence of the internal standard fragment is as shown in SEQ ID NO: shown at 10.
7. The kit according to claim 6, wherein the sequences of the primer group of the amplified internal standard fragment are shown in SEQ ID NO: 11 and 12, the sequence of the detection probe is shown as SEQ ID NO: shown at 13.
8. The kit according to any one of claims 1 to 7, further comprising at least one of a plasma nucleic acid free DNA extraction reagent, a PCR reaction buffer, deoxyribonucleoside triphosphates, a positive control, a negative control, and an enzyme mixture.
9. The kit of claim 8, wherein the enzyme mixture comprises thermostable DNA polymerase and uracil DNA glycosylase.
10. A method for detecting free nucleic acid of blood stream infection pathogen in plasma uses plasma as a detection sample, uses DNA of the free nucleic acid of the blood stream infection pathogen as a detection marker, and uses a primer group of a target sequence of the DNA of the blood stream infection pathogen and a detection probe to carry out QPCR detection on the free nucleic acid of the blood stream infection pathogen in the plasma.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911221222.5A CN110714090B (en) | 2019-12-03 | 2019-12-03 | Kit for detecting free nucleic acid of blood flow infection pathogen in blood plasma |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911221222.5A CN110714090B (en) | 2019-12-03 | 2019-12-03 | Kit for detecting free nucleic acid of blood flow infection pathogen in blood plasma |
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| Publication Number | Publication Date |
|---|---|
| CN110714090A true CN110714090A (en) | 2020-01-21 |
| CN110714090B CN110714090B (en) | 2023-08-22 |
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| CN111705118A (en) * | 2020-06-23 | 2020-09-25 | 宁夏医科大学总医院 | Blood stream infection detection kit based on target gene high-throughput sequencing |
| CN112575099A (en) * | 2020-12-25 | 2021-03-30 | 郑州安图生物工程股份有限公司 | Primer-probe combination and detection kit for klebsiella pneumoniae |
| CN112813179A (en) * | 2020-12-09 | 2021-05-18 | 广州市第一人民医院（广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院） | Kit for detecting urinary tract infection pathogenic bacteria and use method thereof |
| CN113512602A (en) * | 2021-07-08 | 2021-10-19 | 华东医院 | Blood stream infection pathogen multiple gene detection system and kit and application thereof |
| CN114592080A (en) * | 2022-04-01 | 2022-06-07 | 领航基因科技（杭州）有限公司 | RT-ddPCR reagent for detecting four pathogenic bacteria |
| CN114622021A (en) * | 2020-12-11 | 2022-06-14 | 张祥林 | Kit suitable for simultaneously detecting eleven pathogenic bacteria and detection method thereof |
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| CN112575099A (en) * | 2020-12-25 | 2021-03-30 | 郑州安图生物工程股份有限公司 | Primer-probe combination and detection kit for klebsiella pneumoniae |
| CN112575099B (en) * | 2020-12-25 | 2023-11-10 | 郑州安图生物工程股份有限公司 | Primer probe combination and detection kit for klebsiella pneumoniae |
| CN113512602A (en) * | 2021-07-08 | 2021-10-19 | 华东医院 | Blood stream infection pathogen multiple gene detection system and kit and application thereof |
| CN113512602B (en) * | 2021-07-08 | 2022-10-04 | 华东医院 | Blood stream infection pathogen multiple gene detection system and kit and application thereof |
| CN114592080A (en) * | 2022-04-01 | 2022-06-07 | 领航基因科技（杭州）有限公司 | RT-ddPCR reagent for detecting four pathogenic bacteria |
| CN114592080B (en) * | 2022-04-01 | 2023-12-01 | 领航基因科技（杭州）有限公司 | RT-ddPCR reagent for detecting four pathogenic bacteria |
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